Project Summary The current project proposes an original framework to translate basic mouse immunology of the pre-clinical phase of type 1 diabetes (T1D) to human, using sophisticated single cell technologies. Over the past four years, we have dissected the antigen-specific CD4+ T cell response in the NOD mouse model using single cell gene profiling, TCR sequencing, and RNAseq to understand the process of activation of autoreactive T cells in target organs. After defining a unique program of activation for islet-specific CD4+ T cells, we isolated the same cells from peripheral blood of pre-diabetic mice. Our contention is that similar studies can now be done in human and help diagnose disease at a very early stage and follow disease evolution and monitor therapeutic intervention. In addition, the same approach will deliver important mechanistic insights in the role of CD4+ T cells in T1D onset and progression. Our work will be focused on two specific aims: SA #1: Expand the mouse studies to optimize their translational value. In the NOD mouse model, single cell analysis of islet CD4+ T cells has allowed the identification of recirculating autoreactive CD4+ T cells in the peripheral blood. Probing two antigen specificities with a series of pMHC tetramers, the profile of pathogenic cells was dissected by gene expression profiling, RNAseq, and TCR ?? pair sequencing. Using the same approach, further characterization of these cells will allow us to gain mechanistic insights and identify potential new therapeutic targets. We hypothesize that recirculating T cells from all stages of the disease process can be found in blood and analyzed by single cell technologies, and used to diagnose and follow disease evolution. SA #2: Analysis of activated CD4+ T cells from the peripheral blood of T1D patients. Using the same approaches and single cell technology, we will characterize a circulating CD4+HLA-DR+PD-1+CXCR3+ cell population that we have identified in T1D patients and not in controls. Antigen specificity will be examined using HLA-DQ tetramers, and functionally using T cell activation assays after TCR re-expression of sequenced ?? pairs as well as new humanized mouse models. A single antigen, insulin, and common mouse-human epitopes will be used for this translation before additional antigen reactivities are examined. The approach will be tested for its ability to measure the anti-islet autoreactivity in ?at-risk?, ?just-diagnosed?, and ?established? T1D patients, and compared to the classic anti-islet antibody detection. The two aims will also evaluate the precise role of the ?P9 switch? mode of T cell recognition in mice and human T1D, respectively, and potentially answer why this disease is linked to the single HLA class II ?57 polymorphism. Hopefully, we will demonstrate that single cell technologies by interrogating rare cells in peripheral blood, have the power to diagnose T1D in its pre-clinical phase in at risk patients. Such a revolutionary approach would permit to monitor evolution and new therapies aimed at maintaining ? cell mass.